Methods and products for production of wax esters (u.s. national phase)

Abstract

The present invention relates to the provision of genetically modified fungal cells, such as yeast cells with an improved ability for producing different fatty acids and specifically fatty acid ethyl esters (FAEE), the main components of biodiesel. An increased in fatty acid production, and hence in FAEE, is obtained in the first place by expressing different heterologous polypeptides in combination with the down-regulation, attenuation, deletion or over-expression of specially selected genes, wherein said genes encode enzymes involved in the fatty acids synthesizing pathway, fatty acid consuming pathways, carbohydrate biosynthesis pathways or enzyme acting as wax ester transporters or a combination thereof. The methods and products of the invention would allow large-scale production of FAEE with carbohydrates as the only externally-supplied substrate.

1 . A fungal cell system for producing fatty acyl ethyl esters (FAEE), said system comprising:
 a) a fungal cell, and   b) an expression vector encoding at least one wax synthase,   wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester transporter(s) of said fungal cell.   
     
     
         2 . A fungal cell system according to  claim 1 , wherein said fungal cell is a yeast cell. 
     
     
         3 . A fungal cell system according to  claim 1 , wherein said fungal cell is selected from the group of fungal cells consisting of  Saccharomyces, Saccharomyces cerevisae Hansenula polymorpha, Kluyveromyces, Pichia, Candida albicans, Aspergilli, Rhodotorula rubra, Torulopsis, Trichosporon cutaneum, Trichoderma reesei, Apiofrichum curvafum, Yarrowia lipolytica , and  Cryptococcus curvatus.    
     
     
         4 . A fungal cell system according to any one of  claims 1 - 3 , wherein said modification is genetic and is effectuated by the introduction of one or more exogenous expression vector(s) into said fungal cell. 
     
     
         5 . A fungal cell system according to  claim 4 , wherein said one or more exogenous expression vector(s) is a plasmid. 
     
     
         6 . A fungal cell system according to any one of  claims 1 - 5 , wherein said genetic modification provides for an increased supply of fatty acyls to the metabolism of said fungal cell. 
     
     
         7 . A fungal cell system according to any one of  claims 1 - 6 , wherein said fungal cell is genetically modified to stimulate overproduction of fatty acids. 
     
     
         8 . A fungal cell system according to any one of  claims 1 - 7 , wherein the modification is performed to any one or more of the following genes, or its expression products: ACB1 (ACBP, acyl-CoA-binding protein), ACC1 (Acetyl-CoA carboxylase), FAS1, FAS2 (Fatty acid synthase), gapN (NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase), ACS1 (Acetyl-CoA synthetase), DGA1 (Acyl-CoA:diacylglycerol acyltransferas), LRO1 (Lecithin: cholesterol acyltransferase), ARE1, ARE2 (Acyl-CoA:sterol acyltransferase), PDX1 (Peroxisomal acyl-CoA oxidase). 
     
     
         9 . A fungal cell system according to  claim 8 , wherein the modification is performed by a knockout/deletion of one or more of the genes DGA1, LRO1, ARE1, ARE2, and PDX1. 
     
     
         10 . A fungal cell system according to  claim 8 , wherein the modification is performed by overexpressing one or more gene product(s) by the introduction of one or more expression vector(s) encoding said one or more gene product(s), said one or more gene product(s) being selected from the group consisting of: acyl-CoA-binding protein, Acetyl-CoA carboxylase (ACC1), NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase, Fatty acid synthases (FAS1, FAS2) and Acetyl-CoA synthetase (ACS1). 
     
     
         11 . A fungal cell system according to  claim 10 , wherein said modification provides for an overexpression of ACC1 in combination with an increased expression of FAS1 and FAS2. 
     
     
         12 . A fungal cell system according to any one of  claim 10  or  11 , wherein the modification of ACC1 is performed by the introduction of an expression vector and an increased expression of FAS1/FAS2 is performed by replacing the promoter thereof. 
     
     
         13 . A fungal cell system according to  claim 12 , wherein Ser659Ala and Ser1157Ala of said ACC1 gene is replaced (SEQ ID NO:16). 
     
     
         14 . A fungal cell system according to any one of the preceding claims, wherein said wax synthase encoded by said expression vector is heterologous. 
     
     
         15 . A fungal cell system according to any one of the preceding claims, wherein said wax synthase is obtained from the one or more of the species  Mycobacterium, Rhodococcus, Acinetobacter, Mus Musculus  and/or  Marinobacter.    
     
     
         16 . A fungal cell system according to  claim 15 , wherein said at least one wax synthase is selected from the group consisting of  Acinetobacter baylyi  ADP1,  Marinobacter hydrocarbonoclasticus  DSM 8798,  Rhodococcus opacus  PD630,  Mus musculus  C57BL/6, and  Psychrobacter articus  273-4. 
     
     
         17 . A fungal cell system according to  claim 16 , wherein the gene expressing said wax synthase is codon optimized and comprises a sequence encoded by any one of SEQ ID NO:1, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and/or SEQ ID NO 7. 
     
     
         18 . A fungal cell system according to  claim 16  or  17 , wherein said wax synthase is encoded by one or more of the following expression vectors pSP-B1, pSP-B2, pSP-B3, pSP-B4 and/or pSP-B5. 
     
     
         19 . A fungal cell system according to any one of the preceding claims, wherein said expression vector encoding said one or more wax synthase(s) is an episomal plasmid (single copy plasmids) or a high-copy plasmid. 
     
     
         20 . A fungal cell system according to any one of the preceding claims, wherein said expression vector encoding said wax synthase provides for chromosomal integration into the chromosome of said fungal cell. 
     
     
         21 . A fungal cell system according to any one of the preceding claims, wherein carbohydrates are supplied as an external substrate to said fungal cell system for the production of FAEE. 
     
     
         22 . A fungal cell system according to  claim 21 , wherein said carbohydrates are selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose. 
     
     
         23 . A fungal cell system according to any one of the preceding claims, wherein additionally either or both of genes Eht1p and Eeb1p of said fungal cell, are overexpressed by said fungal cell. 
     
     
         24 . Use of an fatty acyl ester, such as a fatty acyl ethyl ester (FAEE), produced by a fungal cell system according to any one of the preceding claims as a component in a biofuel, such as biodiesel, a lubricant, cosmetic, linoleum, printing ink, and/or a solid wax ester used for candles and/or polishes. 
     
     
         25 . A composition comprising a fungal cell system according to any one of the preceding claims, said composition further comprising at least one additional component selected from the group consisting of: buffers; stabilizers; protease-inhibiting agents; hydrolytic enzymes, saccharolytic enzymes; cell membrane- and/or cell wall-preserving compounds, nutritional media appropriate to the cell; and the like. 
     
     
         26 . A method for producing fatty acyl ethyl esters (FAEE), said method comprising:
 a) providing a fungal cell system according to any one of  claims 1 - 23  and,   b) adding one or more sources of carbohydrates as an external substrate to said fungal cell system in a culture broth;   c) and wherein said FAEE are thereafter retrieved by extraction from said culture broth.   
     
     
         27 . A method according to  claim 26 , wherein said carbohydrates are selected from the group consisting of: glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, cellulose, and hemicellulose. 
     
     
         28 . A composition comprising:
 a) a fungal cell which metabolism is modified thereby possessing an increased flux towards fatty acid biosynthesis; and   b) one or more expression vectors encoding one or more wax synthase(s)   
     
     
         29 . Use of a composition according to  claim 28  for producing biofuel esters, such as biodiesel, lubricants, cosmetics, linoleum and printing inks, and/or the solid waxes used for candles and polishes. 
     
     
         30 . A yeast cell having an increased metabolic flux towards fatty acid ester biosynthesis, said yeast cell expressing at least one wax synthase selected from the group consisting of  Acinetobacter baylyi  ADP1,  Marinobacter hydrocarbonoclasticus  DSM 8798,  Rhodococcus opacus  PD630,  Mus musculus  C57BL/6 and  Psychrobacter articus  273-4) in combination with over-expressing the protein ACBP (acyl-CoA-binding protein) 
     
     
         31 . A yeast cell according to  claim 30 , wherein said yeast cell is  Saccharomyces cerevisae.    
     
     
         32 . A fungal cell system for overproducing fatty acids, comprising a fungal cell having an increased metabolic flux towards fatty acid biosynthesis, characterized by at least over-expressing ACC1 (Acetyl-CoA carboxylase). 
     
     
         33 . A fungal cell system for overproducing fatty acids, comprising
 a) a fungal cell having an increased metabolic flux towards fatty acid biosynthesis, characterized by at least over-expressing ACC1 (Acetyl-CoA carboxylase), and wherein the metabolism of said fungal cell is additionally modified, said modification providing for down-regulation, attenuation, deletion and/or over-expression of one or more gene(s) selected from the group consisting of genes encoding one or more enzyme(s) involved in at least one of said fungal cell's fatty acid synthesizing pathways, fatty acid consuming pathways and carbohydrate biosynthesis pathways, and/or selected from the group consisting of genes encoding one or more enzyme(s) acting as wax ester and/or fatty acids transporter(s) of said fungal cell.